Arachidonic acid releasing phospholipase A2 enzyme and methods of use

Abstract

The invention provides novel DNA and peptide sequences encoding a family of phospholipase A 2 enzymes, with specific activities of approximately 20 μmol/min/mg in the mixed micelle assay. These enzymes are useful in methods for detecting the anti-inflammatory potential of various chemical agents. The invention also details novel methods for determining such potential using the novel sequences, methods for making the novel peptides, and methods for developing new anti-inflammatory drugs.

What is claimed is: 
     
       1. In a method of identifying an inhibitor of phospholipase A 2  enzyme including the steps of combining a phospholipid, a candidate inhibitor compound, and a phosphalipase A 2  enzyme and observing whether said phospholipase A 2  enzyme cleaves said phospholipid and releases fatty acid thereby, the improvement comprising: said phospholipase A 2  enzyme being substantially free from association with other proteinaceous materials and being characterized by an amino acid sequence selected from the group consisting of: (a) an amino acid sequence encoded by the DNA sequence of Table I or Table II;   (b) an amino acid sequence encoded by a DNA sequence capable of hybridizing to said DNA sequence of Table I or Table lI under stringent conditions and which is a natural mammalian homologue of said DNA sequence of Table I or Table II; and exhibiting phospholipase A 2  activity in the mixed micelle assay; and   (c) an amino acid sequence encoded by an allelic variant of said DNA sequence of Table I or Table II.     
     
     
       2. The improvement of claim 1 wherein said phospholipase A 2  enzyme is characterized by an amino acid sequence encoded by the DNA sequence of Table I. 
     
     
       3. The improvement of claim 1 wherein said phospholipase A 2  enzyme is characterized by an amino acid sequence encoded by the DNA sequence of Table II. 
     
     
       4. The improvement of claim 1 wherein said phospholipase A 2  enzyme is characterized by an amino acid sequence capable of hybridizing to said DNA sequence of Table I or Table II under stringent conditions and which is a natural mammalian homologue of said DNA sequence of Table I or Table II; and exhibiting phospholipase A 2  activity in the mixed micelle assay. 
     
     
       5. The improvement of claim 1, 2, 3, or 4 wherein the source of phospholipid comprises whole cells. 
     
     
       6. In a method of identifying an inhibitor of phospholipase A 2  enzyme including the steps of combining a phospholipid, a candidate inhibitor compound, and a phospholipase A 2  enzyme and observing whether said phospholipase A 2  enzyme cleaves said phospholipid and releases fatty acid thereby, the improvement comprising: said phospholipase A 2  enzyme being characterized by an amino acid sequence encoded by the DNA sequence of Table I.   
     
     
       7. The improvement of claim 6 wherein the source of phospholipid comprises whole cells.